Bile acid synthesis in cultured human hepatocytes: Support for an alternative biosynthetic pathway to cholic acid

The biosynthesis of bile acids by primary cultures of normal human hepatocy tes has been investigated. A general and sensitive method for the isolation and analysis of sterols and bile acids was used, based on anion exchange c hromatography and gas chromatography-mass spectrometry (GC/MS). Following i ncubation for 5 days, 8 oxysterols and 8 C-27- or C-24-bile acids were iden tified in media and cells. Cholic and chenodeoxycholic acids conjugated wit h glycine or taurine were by far the major steroids found, accounting for 7 0% and 24% of the total, respectively, being consistent with bile acid synt hesis in human liver. Small amounts of sulfated 3 beta-hydroxy-5-cholenoic acid and 3 beta,7 alpha-dihydroxy-5 beta -cholanoic acid were also detected . Nine steroids were potential bile acid precursors (2% of total), the majo r precursors being 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid and it s 5 beta-reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7 alpha-hydroxylation of cholesterol, followed by oxidation to 7 alpha-hydro xy-4-cholesten-3-one and 12 alpha-hydroxylation. Notably, 27-hydroxylation of the product 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one and further oxi dation and cleavage of the side chain precede A-ring reduction. A-Ring redu ction may also occur before side-chain cleavage, but after 27-hydroxylation , yielding 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid as an intermediate. The amounts of the intermediates increased in parallel to those of CA during 4 days of incubation. Suppressing 27-hydroxylation with cyclosporin A (CsA) resulted in a 10-fold accumulation of 7 alpha,12 alpha- dihydroxy-4-cholesten-3- one and a decrease of the production of CA and its acidic precursors. These results suggest that the observed intermediates r eflect an alternative biosynthetic pathway to CA, which may be quantitative ly significant in the cells.